Cancer treatment is entering an era of targeted approaches. One such approach is use of the immune system to recognize and eliminate malignant cells. Synthetic CpG oligonucleotides (CpG DNA) are a relatively new class of agents that have the ability to stimulate a potent, orchestrated tumour-specific immune response (KRIEG, A M. Lymphocyte activation by CpG dinucleotide motifs in prokaryotic DNA. Trends Microbiol. 1996, vol. 4, no. 2, p. 73-6; and KRIEG, A M, et al. Mechanisms and therapeutic applications of immune stimulatory CpG DNA. Pharmacol Ther. 1999, vol. 84, no. 2, p. 113-20.).
Recent studies demonstrate that at least three classes of CpG DNA sequences exist, each with different physical characteristics and biological effects. Preliminary studies in several animal models of cancer suggest that CpG DNA may have many uses in cancer immunotherapy. CpG DNA have the ability to induce tumour regression by activating innate immunity, enhancing antibody dependent cellular cytotoxicity, and serving as potent vaccine adjuvants that elicit a specific, protective immune response. Early clinical trials indicate that CpG DNA can be administered safely to humans, and studies are ongoing to understand how these agents may play a role in cancer immunotherapy (WOOLDRIDGE, J E, et al. CpG DNA and cancer immunotherapy: orchestrating the antitumour immune response. Curr Opin Oncol. 2003 November, vol. 15, no. 6, p. 440-5.)
An early patent (U.S. Pat. No. 6,498,147 B, THE SCRIPPS INSTITUTE, 2002 Dec. 24) presented antisense oligonucleotides and disclosed antisense inhibition of tumour cells in vitro, as well as an animal experiment showing antisense inhibition of tumour growth in vivo in syngenic C57B1/6 mice. The mice were treated with intraperitoneal injections of 40 mg/g sense and antisense oligodeoxynucleotides. Histologic analysis showed focal tumour necrosis followed by widespread segmental necrosis.
B-chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the western world. B-CLL is a cancer of the white blood cells and bone marrow, characterized by uncontrolled proliferation and/or reduced cell death (apoptosis) of blood cells, specifically the B lymphocytes, and is the most widespread form of adult leukemia. Its incidence approaches 50 per 100,000 after the age of seventy. The leukemia usually has a protracted natural course of years and even decades, but eventually accelerates as the cells acquire sequential genetic defects. B-CLL differs from many other malignancies in that monoclonal B-CLL cells accumulate relentlessly, due to an abnormally prolonged life span, which likely is a consequence of altered interactions between defective B-CLL cells and their environment. Cytokines are essential factors in cell homeostasis and cell-cell dialogue, and are proposed to be critical in this milieu (CALIGARIS-CAPPIO et al., 1999, ROZMAN et al., 1995).
No common initial transforming event has been found for B-CLL. Chromosomal translocations, thought to occur mainly during the gene rearrangement process and common in other lymphoid malignancies, are rare in B-CLL. Karyotypic abnormalities tend to increase in frequency and number during the course of the disease. When translocations are found, they tend to result in genetic loss rather than in the formation of a fusion gene or over-expression of an oncogene. The most common genetic abnormalities in B-CLL are 13q deletions (50% of cases), 13q4 deletions (associated with an indolent course), trisomy 12 (12q13-15, with over-expression of the MDMQ oncoprotein which suppresses p53), and 1Iq22-q23 deletions (20% of cases) (GAIDAN et al., 1991 et al., DOHNER et al., 1999).
B-CLL cells express surface molecules such as CD23 (low affinity receptor for IgE), CD25 (IL-2R α chain), and CD27 (co-stimulatory molecule), which in other settings indicate a state of activation. The expression and association of several proteins tightly regulate the process of apoptosis. The relative balance of these proteins controls cell life span. Genes responsible for this system include the BCL-2 family, the tumour necrosis factor receptor and genes such as Myc and p53 (OSORIO et al., 1999). All the death pathways promoted by these genes appear to have a common ‘demolition” cascade, represented by the protease family of the caspases. B-CLL cells consistently express high levels of products of the anti-apoptosis members of the BCL-2 family (bad-2, bcl-n, bax), while the Bcl-2 function inhibitor Bcl-6 is markedly reduced. The mechanism involved in over-expression of Bcl-2 is currently unclear. The leukemic cells of B-CLL are negative or weakly positive for Fas. They generally remain resistant to anti-Fas antibody mediated death even after stimulation induced Fas expression. In rare sensitive cases, cell death occurs independently of Bcl-2 expression by a mechanism still uncharacterized. It would appear that Bcl-2 over-expression and the Fas pathway are mechanisms involved in the pathophysiology of B-CLL but not necessarily critical causative events. Mediators including cytokines are likely to link the initial etiologic factor with the terminal pathways of apoptosis.
Most B-CLL cells are the in G0 phase of the cell cycle and can not be induced to enter the proliferative phase by conventional methods such as concanavalin-A, phorbolesters, or receptor cross-linking, which induce the proliferation of normal lymphocytes. Only a small subset of cells appears to enlarge the clonal population in response to an unknown promoting signal. Proliferation promoting cytokines may provide this stimulus in vivo (DANCESCU et al, 1992).
B-CLL cells accumulate at the expense of the normal B-cell pool. Total T-cells on the other hand, are usually increased. The bone marrow T-lymphocytes are predominantly CD4+ cells as seen in autoimmune disorders such as rheumatoid arthritis and sarcoidosis. There is frequently a Th2 predominant cytokine phenotype in peripheral blood. Abnormalities in the TCR repertoire have been reported also. Reports indicate that T-lymphocytes and stromal cells may have a key role in supporting an environment capable of perpetuating the life span of the B-CLL cells. Both the malignant cells and their T-cell entourage express a vanity of surface molecules and their receptors: CD5 and its ligand CD72, CD27 and CD70. These findings open various possibilities of mutual interaction which could result directly or indirectly (cytokines) in cell self-preservation. Such lengthy survival would, in turn increase chances for accumulation of gene mutations and genetic instability, which favours disease progression through dysregulation of cell cycle check-points, and resistance to cytotoxic therapy (KLEIN et al., 2000).
The symbiotic interaction between B-CLL cells and their environment is almost certainly mediated by the secretion of cytokines and modulated by adhesion molecules. Investigation of cytokine involvement in B-CLL has generated a substantial body of data supporting or disproving various cytokines as mediators of proliferation and prolonged life span in this leukemia. Cytokine production investigations have demonstrated reverse-transcription polymerase chain reaction signals for IL1, IL2, IL3, IL4, IL5, IL7, TNF-β, and TNF-α (PISTOIA et al., 1997). These findings have been contradicted by other studies which showed negative results for IL4, IL3 and IL6 (TANGYE et al., 1999). In contrast, TGF-β, as well as IL10 secretion, has been show in normal B-lymphocytes. No other cytokine production has been reported to be constitutive for these cells.
Immunotherapy of cancer has been explored for over a century, but it is only in the last decade that various antibody-based products have been introduced into the management of patients with diverse forms of cancer. At present, this is one of the most active areas of clinical research, with eight therapeutic products already approved in oncology. Antibodies against tumour-associated markers have been a part of medical practice in immunohistology and in vitro immunoassays for several decades, and are now becoming increasingly recognized as important biological agents for the detection and treatment of cancer (STROME et al., 2007). Molecular engineering has improved the prospects for such antibody-based therapeutics, resulting in different constructs and humanized or human antibodies that can be frequently administered.
CD20 is variably expressed on the surface of B-cells in CLL patients with some patient's B-cells expressing very low levels of CD20 antigen. CD20 (human B-lymphocyte restricted differentiation antigen), is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. The antigen is also expressed on more than 90% of B-cells in non Hodgkin's lymphomas (NHL), but is not found on hematopoietic stem cells, pro B cells, normal plasma cells or other normal tissues. CD20 regulates an early step(s) in the activation process for cell cycle initiation and differentiation, and possibly functions as a calcium ion channel. CD20 is not shed from the cell surface and does not internalize upon antibody binding. Free CD20 antigen is not found in the circulation (PESCOVITZ, 2006).
The anti-CD20 antibody Rituximab, which is a genetically engineered chimeric murine/human monoclonal antibody directed against human CD20 (Rituxan® or MabThera®, from Genentech, Inc., South San Francisco, Calif., U.S.) is used for the treatment of patients with relapsed or refractory low-grade or follicular, CD20 positive, B-cell non-Hodgkin's lymphoma and B-CLL. Rituximab works by recruiting the body's natural defense to attack and kill the B-cell to which it binds via the CD20 antigen. In vitro mechanism of action studies have demonstrated that Rituximab binds human complement and lyses lymphoid B-cell lines through complement-dependent cytotoxicity (CDC) (REFF et al., 1994). Additionally, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). In vivo preclinical studies have shown that Rituximab depletes B-cells from the peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, presumably through complement and cell-mediated processes (REFF et al., 1994). While Rituximab has been used with some success in CLL patients, analysis of CLL patients shows that the density of CD20 on the surface of B-CLL cells is rather variable with some patient's B cells expressing very low levels of CD20 antigen. The typical treatment for B-cell malignancies, besides Rituximab, is the administration of radiation therapy and chemotherapeutic agents. In the case of CLL, conventional external radiation therapy will be used to destroy malignant cells. However, side effects are a limiting factor in this treatment. Another widely used treatment for haematological malignancies is chemotherapy. Combination chemotherapy has some success in reaching partial or complete remissions. Unfortunately, these remissions obtained through chemotherapy are often not durable.
Conversely, CD23 expression has been found to be consistently present at higher levels in B-CLL. The CD23 leukocyte differentiation antigen is a 45 kD type II transmembrane glycoprotein expressed on several haematopoietic lineage cells, which function as a low affinity receptor for IgE (FcyRII) (PATHAN et al., 2008). It is a member of the C-type lectin family and contains an α-helical coiled-coil stalk between the extracellular lectin binding domain and the transmembrane region. The stalk structure is believed to contribute to the oligomerization of membrane-bound CD23 to a trimer during binding to its ligand (for example, IgE). Upon proteolysis, the membrane bound CD23 gives rise to several soluble CD23 (sCD23) molecular weight species (37 kD, 29 kD and 16 kD). In addition to being involved in regulating the production of IgE, CD23 has also been speculated to promote survival of germinal center B cells. The expression of CD23 is highly up-regulated in normal activated follicular B cells and in B-CLL cells.
Lumiliximab is a monoclonal chimeric anti-CD23 antibody (from Biogen Idec, currently undergoing clinical trials) that harbours macaque variable regions and human constant regions (IgG1, κ) and was originally developed to inhibit the production of IgE by activated human blood B-cells. It is now in a Phase III trial for use in B-CLL patients. In vitro studies have shown that Lumiliximab induces caspase dependent apoptosis in B-CLL cells through the mitochondrial death pathway (PATHAN et al., 2008). Thus, it seems to induce apoptosis of tumour cells through a mechanism different from Rituximab.
Several other antibodies have recently been approved for the treatment of cancer. Alemtuzumab (Campath® or MabCampath®, an anti-CD52 from Ilex Pharmaceuticals) (KEATING et al., 2002) was approved in 2001 for the treatment of CLL. Bevacizumab (Avastin®, Genentech, Inc., South San Francisco, Calif.) is a humanized IgG1 mAb directed against vascular endothelial growth factor (VEGF) used in treatment of colorectal cancer, small cell lung cancer and breast cancer. Trastuzumab (Herceptin® from Roche) is a humanized IgG1 mAb that is effective against metastatic breast cancer tumours over-expressing the HER-2 target (STROME et al., 2007).
In order to make antibody drugs more efficient, an up-regulation of the specific antigen targets on the surface of tumour cells might be helpful. One way of obtaining such an effect could be to stimulate the cells with immunomodulatory oligonucleotides. Immune stimulatory effects can be obtained through the use of synthetic DNA-based oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. Such CpG ODN have highly immunostimulatory effects on human and murine leukocytes, inducing B cell proliferation; cytokine and immunoglobulin secretion; natural killer (NK) cell lytic activity and IFN-gamma secretion; and activation of dendritic cells (DCs) and other antigen presenting cells to express co-stimulatory molecules and secrete cytokines, especially the Th1-like cytokines that are important in promoting the development of Th1-like T cell responses (KRIEG et al, 1995). The increase in receptor density by CpG-ODNs could be mediated through a direct effect of the oligonucleotides on the cells, or through the induction of cytokines. An increase in antigen density or an increase in the population of cells expressing the target receptors would enable the antibodies to kill the tumour cells more efficiently, either through enhancing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). WO 95/35032 concerns oligonucleotides (antisense) which hybridize to NF-kB mRNA and methods of using these to suppress processes which depend upon activation of NF-kB. Such processes are typically associated with such disorders as those mediated by immune or cytokine responses (for example, septic or non-septic shock) as well as those disorders induced by infectious agents such as retroviruses, more specifically, HIV and HTLV.
There are indications that the CpG motif alone is not accountable for the efficacy of the oligonucleotides. There are even indications that this motif is not necessary for the desired function.
Regardless of the considerable effort spent on developing oligonucleotide based therapeutic approaches to cancer, and the occasional success reported so far, there still remains a need for new compounds and modes of administration, exhibiting improved efficacy and minimal or no side effects.